Molecular Biotechnology Exam 3 questions with correct answers

Determining the sequence of DNA CORRECT ANSWER Methods: - Chain termination or dideoxy method --- Developed by F. Sanger --- Not used much anymore - Shotgun sequence method - 2nd generation sequence methods --- Pyrosequencing Sanger (dideoxy) method for determining DNA sequence CORRECT ANSWER 1) Denaturation of DNA 2) Primer attachment and extension of bases 3) Termination due to incorporation of dideoxy ribonucleotides 4) Gel electrophoresis to compare the products of extension - The sequence is read from the bottom of the gel, moving up because the fragments at the bottom are the shorter ones. - Ethidium bromide used for visualizing the DNA fragments. Automated version of the dideoxy method of DNA sequencing CORRECT ANSWER Follow the same steps as traditional dideoxy method Each dideoxy ribonucleotide is attached to a different fluorescent dye, so laser is used on the gel, and the reflected light goes through a spinning filter tube with four different filters. Each filter only allows certain colors to go through. When the light is able to go through the spinning filter tube, it will reach a photomultiplier detector tube, which will send data to the computer. The computer software analyses the DNA sequence based on the fluorescence. Problems with the dideoxy method CORRECT ANSWER Goo